Egg albumen was hydrolyzed by proteolytic enzyme, and its antibacterial property was studied by the paper disc method. Egg albumen and its enzymatic hydrolysate formed an inhibition zone for Bacillus subtilis and Streptococcus lactis. The antibacterial property of hydrolyzed egg albumen varied with the degree of hydrolysis and increased two-fold of egg albumen by bromelin or trypsin treatment. Pronase E produced a large quantity of soluble or formol nitrogen, but its hydrolysate had low antibacterial property. The hydrolyzed egg albumen did not have antibacterial properties for B. cereus and B. cereus var. mycoides, but had antibacterial property for B. coagulans and B. licheniformis.

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Strains of Proteus vulgaris utilized only citric acid among the tricarboxylic acids. Citric acid uptake in P. vulgaris IFO 3045 was mediated by a single transport system induced by citrate, isocitrate or cis-aconitate and possessed a weak affinity to isocitric acid. This citric acid transport system corresponds to the system of Salmonella typhimurium or Serratia marcescens, which is induced by citrate, isocitrate or cis-aconitate and transports citric and isocitric acids.

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Among 920 strains of fungi tested only black koji-molds were found to be potent producers of 5'-phosphodiesterase. The enzyme from Aspergillus niger was partially purified from aqueous extract of the culture on wheat bran. The enzyme hydrolyzed 3'-hydroxyl-terminated 3'→5' and 2'→5' dinucleotides or 3'→5' and 2'→5' dinucleoside monophosphates without specificity toward bases or sugars. The enzyme also hydrolyzed p-nitrophenyl ester of nucleoside 5'-monophosphate, ADP, CDP-choline and UDPG to give nucleoside 5'-monopho-sphate. The enzyme was actually proved to split 2'→5' dinucleotides in nuclease P₁ digest of technical grade yeast RNA containing several per cent of 2'-5' phosphodiester linkages in addition to 3'-5' linkages.

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A new protease, Sfericase, found in culture filtrate of a strain of Bacillus sphaericus was purified and isolated in a crystalline form by salting out with ammonium sulfate followed by column chromatography on DEAE-Sephadex A-50 (using a specific elution buffer). The optimal and maximal caseinolytic activities of the protease were around pH 9.0_??_9.3 and at 55°C, respectively. The proteolytic activity was inhibited by DFP, potato protease inhibitors and EDTA, but not by PCMB, o-phenanthroline, TPCK and TLCK. The molecular weight of the protease was estimated by sedimentation equilibrium (32, 000±400) and gel chromatography (27, 000).
The enzyme molecule contained two atoms of undialyzable calcium ion and two halfcystine residues which should afford one disulfide linkage as no free sulfhydryl group was found even with the denatured enzyme.
By immunological cross reaction, it was indicated that the enzyme was distinct from other alkaline proteases of Bacillus origins.

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Penicillium oxalicum produced two forms (isoenzymes) of glucoamylase which could be separated from each other by gel electrofocusing, and they were designated as glucoamylase I and glucoamylase II. Glucoamylases I and II were homogeneous on polyacrylamide gel electrophoresis, gel electrofocusing and ultracentrifugation, respectively. The sedimentation constant (s⁰₂₀, _w) and molecular weight of glucoamylase I were 4.42 S and 84, 000, and those of glucoamylase II were 4.53 S and 86, 000, respectively. Certain properties of these enzymes were also investigated.

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Peptidoglycan portion was isolated from the hydrolysate of Streptomyces roseochromogenes IAM 53 cell walls after hydrolysis with egg white lysozyme. Further hydrolysis by the Flavobacterium lytic enzyme gave rise to a disaccharide and two types of peptides. The disaccharide was β-1, 4-N-acetylglucosaminyl-N-acetylmuramic acid. The main type of peptide was L-alanyl-D-isoglutaminyl-(glycyl-) LL-diaminopimelyl-D-alanine, and D-alanine was missing in the minor type of peptide. Edman degradation and partial hydrolysis of the peptide oligomer revealed the major type of peptidoglycan which was characterized by the presence of LL-diaminopimelic acid and cross-linkage of single glycine. Other species of Streptomyces seemed to have the same type of peptidoglycan as that of S. roseochromogenes.

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The polysaccharide-peptidoglycan complex, which was prepared with lysozyme from Streptomyces roseochromogenes IAM 53 cell walls, was hydrolyzed with lytic enzyme of Flavobacterium to separate polysaccharide. The enzymatically prepared polysaccharide (100mg) contained 500 μmoles of hexoses, 40 μmoles of hexosamines and 31 μmoles of phosphate. Hexoses consisted of mannose and galactose in a molar ratio of 5 to 1. Hexosamines consisted of equimolar glucosamine and muramic acid, a half of which was identified as muramic acid 6-phosphate. The reducing end of the polysaccharide was muramic acid. The polysaccharide extracted with trichloroacetic acid contained no muramic acid-phosphate. So the polysaccharide moiety of S. roseochromogenes cell walls must be linked covalently to 6-position of muramic acid in peptidoglycan through phosphate,

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Analysis methods of a herbicide “bromacil” were studied comparatively in soil and mandarin orange fruit. Bromacil content in tissue of mouse was also measured. Measurement was performed by gas chromatography with electron capture detector (ECD). Mass fragmentography was also studied, which was found to be useful for the residue analysis of this herbicide.

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Four enzymes which transferred amino group of L-alanine to α-keto acids were separated from Pseudomonas sp. and these enzymes were distinguished in substrate specificities from each other: the first is active on glyoxylate; the second, on straight-chain α-keto acids, e. g., α-ketobutyrate, α-ketovalerate and α-ketocaproate; the third, on various α-keto acids including α-ketoglutarate. The fourth is also active on various α-keto acids, however not active on α-ketoglutarate.

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The effect of pH in the SDS-phenol extraction step on the extractability, messenger activity and the composition of RNA species of the poly (A)-containing RNA from the cotyledons of soybean seeds was examined. The amount of total RNA extracted from the cotyledons was almost the same under neutral and alkaline pH. However the content of the poly (A)-containing RNA was higher in the preparation obtained under alkaline pH than neutral pH. higher in the preparation obtained under alkaline pH than neutral pH. This result shows that the poly (A)-containing RNA was efficiently extracted from soybean seeds under alkaline pH. In a mRNA-dependent cell-free system derived from E. coli, there was no significant difference in the messenger activity of the poly (A)-containing RNA between the two preparations. The patterns of the poly (A)-containing RNAs of the two preparations were very similar to one another in the case of dry soybean seeds, while there was slight difference in the case of green soybean seeds. The poly (A)-containing RNA obtained under alkaline pH contained a higher ratio of large RNA species than that obtained under neutral pH. On the basis of these results, for a more efficient recovery of poly (A)-containing RNA, it seems better to employ alkaline pH in the phenol-extraction step for preparing poly (A)-containing RNA from soybean seeds.

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Theaflavins, the oxidized products formed from tea leaf catechins during black tea fermentation, showed an antiviral activity on TMV. From the survey of the interactions of theaflavins with RNA and its related substances, it was presumed that theaflavins disturbed the replication cycle of TMV through binding to TMV-RNA.

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Isocitrate lyase was purified partially from n-alkane-grown cells and glucose-grown cells of Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. The preparation from alkane-grown cells showed one peak of the enzyme activity, while that from glucose-grown cells showed two distinct peaks of the activity, on DEAE-cellulose column chromatography. These enzymes, having the similar pH optima (around 7.0) and Km values with DL-isocitrate (1.2_??_1.7mM), were inhibited by various metabolic intermediates, such as 6-phosphogluconate and phosphoenolpyruvate.
Time-course changes in the activities of isocitrate lyase and isocitrate dehydrogenases of C. tropicalis during the growth indicated that the lyase would participate preferentially in alkane assimilation and NAD-linked isocitrate dehydrogenase in glucose utilization of the yeast.
Regulation of isocitrate metabolism in C. tropicalis through glyoxylate cycle and tricarboxylic acid cycle is discussed based on the kinetic properties, cellular localization and timecourse changes in the levels of isocitrate lyase and NAD-linked and NADP-linked isocitrate dehydrogenases.

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Two main steps of photodecomposition were observed at the initial stage on the irradiation of ultraviolet light to Kitazin P^® in a thin film. One was the isomerization to a thionate, O-benzyl O, O-diisopropyl phosphorothionate, which was gradually hydrolyzed or oxidized to its oxygen analogue. The other step was the cleavage of P-S bond to produce O, O-diisopropyl phosphonate and α-toluenethiol, the latter of which was degraded to produce α-toluenesul-fonic acid via dibenzyl disulfide, and finally sulfuric acid and benzoic acid. O, O-Diisopropyl hydrogen phosphorothioate, O, O-diisopropyl hydrogen phosphate and benzyl alcohol were detected as hydrolyzates. Benzyl alcohol was further oxidized to benzoic acid via benzaldehyde. In addition to these compounds, O, O, S-triisopropyl phosphorothiolate, O, O, O-triisopropyl phosphorothionate, O, O, O-triisopropyl phosphate and benzyl isopropyl sulfide were also detected.

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About 30 dipeptides and some tripeptides were led to new benzimidazole derivatives by incorporating their carboxyl groups into benzimidazole ring by the reaction with o-phenylene-diamine. The ring closure to benzimidazole was well achieved by heating mildly at a moderate temperature in acetic acid.
Some benzimidazole derivatives of peptides had remarkable phytotoxicities.

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Bacteria which can hydrolyze DL-5-indolylmethylhydantoin to L-tryptophan were isolated from various soils. The DL-5-indolylmethylhydantoin-hydrolyzing enzymes were found to be inducible and intracellular. With intact cells, 50mg/ml as wet base, of newly isolated bacterial strain T-523, 10mg/ml of DL-5-indolylmethylhydantoin dissapeared and 7.4mg/ml of L-tryptophan in a molar yield of 82% was produced after 35 hr incubation. Tryptophan produced was confirmed to be L-form regardless of the stereoisomer of the substrates used. A mechanism of asymmetric hydrolysis of DL-5-indolylmethylhydantoin was discussed.

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Lipoxygenase (linoleate: oxygen oxidoreductase, EC 1. 13. 1. 13) from tubers of potato (Solanum tuberosum) was purified 210-fold with a specific activity of 25 units/mg protein, using DEAE-Sephadex column chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme was electrophoretically pure. Some properties of this enzyme were the same as those of potato lipoxygenase previously reported.
The enzyme was inhibited by saturated alcohols and SH-reducing reagents as well as soybean lipoxygenase was. The trapped radical of linoleic acid in potato lipoxygenase reaction was detected by ESR spectroscopy, using 2-methyl-2-nitrosopropane as a radical scavenger. Those suggest that the reaction of potato lipoxygenase proceeds by a very similar mechanism to soybean lipoxygenase.

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Renaturation of soybean 11 S globulin was carried out by dialysis against 0.035M phosphate buffer (pH 7.6) containing 0.4M NaCl starting from denatured states in 8M urea solution either in the presence or absence of 0.2M 2-mercaptoethanol.
The yield of the renatured 11 S globulin was 70% from the denatured state as judged by gel filtration. The purified renatured 11 S globulin from the denatured state was found to be identical with the native protein when judged from gel chromatographic, immunological, ultracentrifugal, gel electrophoretic and circular dichroic analyses, as well as by studies of subunit composition and of susceptibility to proteolytic digestion. On the other hand, the renaturation of 11 S globulin from the reductively denatured state yielded a protein which was somewhat different from the native protein as judged from gel electrophoretic and circular dichroic analyses, and also by studies of subunit composition and of susceptibility to proteolytic digestion.

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An active β-amylase was purified from germinated rice seeds by precipitation with ammonium sulfate, acid treatment, chromatographies on DEAE-cellulose and DEAE-Sephadex A-50, and gel filtrations on Sephadex G-75. The purified enzyme was homogeneous in disc electrophoretic analysis.
The molecular weight was estimated to be approximately 53, 000 by thin-layer gel filtration and polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 5.0 by disc electrofocusing.
The optimum pH was found to be in the pH range of 5.5 to 6.5. The Km value for soluble starch was 3mg/ml. The enzyme was inhibited by sulfhydryl reagents or heavy metal ions.
The active β-amylase was oxidatively dimerized by treatment with 0.3M ferricyanide in 3M urea. The dimerized enzyme was thought to be one of inert β-amylases in ungerminated rice seeds.

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During the screening of metabolites of microorganisms for new insecticides, many piericidin-like substances were found in the mycelial extract of Streptomyces pactum. The isolation and physical properties of these piericidins are described.

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Chemical structures of new piericidins produced by Streptomyces pactum are elucidated on the basis of mass, PMR and CMR spectral analyses. Consequently, these piericidins were shown to be constructed by a combination of variations in each four functionalities and carbon skeletons.

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Rapid, convenient and accurate quantitative assay method of D (-) and L (+)-lactate in soy sauce or wine was established by the combination of a new carboxylic acid analyzer and a discrete type auto analyzer with the aid of L (+)-lactate dehydrogenase. This method does not require specific pretreatment of any samples and samples can be analyzed only after the dilution, filtration or deproteinization.

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A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4 B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39, 000. The optimal pH for the enzymic conversion of caffeic acid to deydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C.

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Dehydrodicaffeic acid dilactone (DDACD) was found in a cultured mushroom by screening for catechol-O-methyltransferase inhibitors. The enzyme which converts two molecules of caffeic acid to DDCAD has been extracted from the mushroom and purified and the enzyme reaction has been studied. It was markedly inhibited by reducing agents, such as NADPH, NADH, glutathione and ascorbic acid but stimulated by Fe³⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cu⁺ and Zn²⁺ ions. Sodium diethyldithiocarbamate and sodium cyanide known to be copper chelating agents inactivated the enzyme, but activity was restored by addition of Cu²⁺ or Cu⁺. Although the enzymic reaction did not occur under anaerobic conditions, ¹⁸O-oxygen was not incorporated into DDCAD. o-Diphenol oxidase catalyzed DDCAD formation from caffeic acid and the DDCAD-forming enzyme catalyzed the formation of DOPAchrome from DOPA. Thus, the DDCAD-formingenzyme is a type of o-diphenol oxidase. Peroxidase and hydrogen peroxide produced DDCAD from caffeic acid.
On the other hand, DDCAD was non-enzymatically synthesized from caffeic acid in the presence of CuCl₂ in 64%. yield. In both enzymic and non-enzymic syntheses, both (+)-DDCAD and (-)-DDCAD were produced.

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The degradation of zein during germination of corn was investigated in special relevance to protease activity. Polyacrylamide gel electrophoresis demonstrated that two main subunits of zein were rapidly degraded as the germination proceeded. It was observed that the zein degradation was followed by the formation of free amino acids, especially phenylalanine and tyrosine. A protease activity increased remarkably during the germination. A crude extract was obtained which could degrade zein in vitro to form relatively large amounts of free phenylalanine and tyrosine. The crude extract showed a maximum activity for casein at pH 3 and for zein at pH 4.5. The activity was enhanced when determined in the presence of 2-mercapto-ethanol. A sulfhydryl protease was estimated to occur which might be responsible for the degradation of zein during germination.

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A protease occurring in the endosperm fraction of germinating corn was purified by means of (NH₄)₂SO₄ fractionation, CM-cellulose chromatography, DEAE-cellulose chromatography, Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified protease was found to have a molecular weight of about 21, 000 and an isoelectric point of pH 2.3 or lower. The optimum pH was found to lie at 3.0 when measured with denatured hemoglobin as substrate. The protease was generally activated by thiol compounds and completely inhibited by p-chloromercuribenzoic acid. Neither diisopropylphosphofluoridate nor diazoacetyl-DL-norleucine methyl ester affected the protease activity. Antipain greatly inhibited the protease action whereas pepstatin had no significant effect. These data indicate, in conclusion, that the protease possesses a unique property to be a sulfhydryl enzyme most active in an acidic region around pH 3.

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